The Drosophila membrane-associated guanylate kinase (MAGUK) protein Polychaetoid (Pyd) is required for dorsal closure of the embryo, sensory organ patterning, and cell fate specification in the developing eye. We demonstrate that pyd is alternatively spliced resulting in two isoforms that differ by the presence or absence of exon 6. To determine the role of alternative splicing in Pyd function, we generated antibodies specific for each isoform. We find that the exon 6(+) form of Pyd is localized at adherens junctions of embryonic and imaginal epithelia, while the exon 6(-) form is distributed broadly along the lateral membrane. These results suggest that localization of Pyd is controlled by alternative splicing and raise the possibility that exon 6 represents a distinct protein-protein interaction domain.